For reconstitution add 90 µl of milliQ water. Please notice that this product contains 10% glycerol and might appear as liquid but is provided lyophilized.

Storage

Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

The AtpB protein standard can be used in combination with global anti-AtpB antibodies to quantitate AtpB from a wide range of species. Global antibodies are raised against highly conserved amino acid sequences in the AtpB protein.

Standard curve: 3 loads are recommended (0.5, 2 and 4μl).For most applications a sample load of 0.2μg of chlorophyll will give a AtpB signal in this range.

Positive control: load per well: a 2μl load is optimal for most chemiluminescent detection systems.

This standard is stabilized and ready and does not require heating before loading on the gel.Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

Expected | apparent MW

in most gel systems AtpB migrates around 50-54 kDa

Confirmed reactivity

Predicted reactivity

Not reactive in

Additional information

Concentration: after adding 90 µl of dest. water final concentration of the standard is 0.27 pmol/µl.

This standard is ready-to-load and does not require any additions or heating. It needs to be fully thawed and thoroughly mixed prior to using. Avoid vigorous vortexing, as buffers contain detergent. Following mixing, briefly pulse in a microcentrifuge to collect material from cap.This standard is stabilized and ready and does not require heating before loading on the gel. Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

AtpB protein standard (AS03 030S) 0.03, 0.1, 0.26 pmol (1-3) and total proteinfrom Trichodesmium IMS 101 extracted with PEB (AS08 300) were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 50 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-hen IgY horseradish peroxidase conjugated) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according the manufacturer's instructions. Images of the blots were obtained using a CCD imager nd Quantity One software Exposure time was 10 seconds. Note: Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.

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